Biology subjects

Ozer, A.

Publications and source records attributed to Ozer, A..

13 recordsLinked to original sources

bioRxiv2025

Identification of human RNA Polymerase II interactors at early stages of transcription.

RNA Polymerase II (Pol II) transcription is highly regulated at two early steps in the transcription cycle: Pre-Initiation Complex (PIC) assembly with its coupled initiation, and promoter-proximal pausing with its controlled release. Here, we developed an optimized biochemical purification method that captures endogenously tagged chromatin-bound Pol II complexes under native conditions at these rate-limiting steps. We then identified a large set of Pol II interactors by mass spectrometry and determined the footprints of these assemblies on promoters with high resolution. Many well-known and new or understudied factors were identified as associated with the PIC and promoter-proximal paused complexes, indicating that despite decades of efforts, these rate-limiting steps of the transcription cycle are far from being completely understood. The new and understudied factors implicate novel mechanisms of regulation that will need to be characterized to fully understand Pol II regulation.

molecular biology↗

bioRxiv2025

Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

Enhancers are gene-distal cis-regulatory elements that drive cell type-specific gene expression. While significant progress has been made in identifying enhancers and characterizing their epigenomic features, much less effort has been devoted to elucidating mechanistic interactions among clusters of functionally linked regulatory elements within their endogenous chromatin contexts. Here, we developed a novel recombinase-mediated genome rewriting platform and applied our divergent transcription architectural model to understand how a long-range human enhancer confers a remarkable 10,000-fold activation to its target gene, NMU, at its native locus. Our systematic dissection reveals transcription factor synergy at this enhancer and highlights the interplay between a divergently transcribed core enhancer unit and emerging new types of cis-regulatory elements--notably, intrinsically inactive facilitators that augment and buffer core enhancer activity, and an adjacent retroviral long terminal repeat promoter that represses enhancer activity. We discuss the broader implications of our focused study on enhancer mechanisms and regulation genome-wide.

genomics↗

bioRxiv2025

High-resolution reconstruction of cell-type specific transcriptional regulatory processes from bulk sequencing samples

Biological systems exhibit remarkable heterogeneity, characterized by intricate interplay among diverse cell types. Resolving the regulatory processes of specific cell types is crucial for delineating developmental mechanisms and disease etiologies. While single-cell sequencing methods such as scRNA-seq and scATAC-seq have revolutionized our understanding of individual cellular functions, adapting bulk genome-wide assays to achieve single-cell resolution of other genomic features remains a significant technical challenge. Here, we introduce Deep-learning-based DEconvolution of Tissue profiles with Accurate Interpretation of Locus-specific Signals (DeepDETAILS), a novel quasi-supervised framework to reconstruct cell-type-specific genomic signals with base-pair precision. DeepDETAILS core innovation lies in its ability to perform cross-modality deconvolution using scATAC-seq reference libraries for other bulk datasets, benefiting from the affordability and availability of scATAC-seq data. DeepDETAILS enables high-resolution mapping of genomic signals across diverse cell types, with great versatility for various omics datasets, including nascent transcript sequencing (such as PRO-cap and PRO-seq) and ChIP-seq for chromatin modifications. Our results demonstrate that DeepDETAILS significantly outperformed traditional statistical deconvolution methods. Using DeepDETAILS, we developed a comprehensive compendium of high-resolution nascent transcription and histone modification signals across 39 diverse human tissues and 86 distinct cell types. Furthermore, we applied our compendium to fine-map risk variants associated with Primary Sclerosing Cholangitis (PSC), a progressive cholestatic liver disorder, and revealed a potential etiology of the disease. Our tool and compendium provide invaluable insights into cellular complexity, opening new avenues for studying biological processes in various contexts.

bioinformatics↗

bioRxiv2025

Comprehensive Evaluation of Diverse Massively Parallel Reporter Assays to Functionally Characterize Human Enhancers Genome-wide

Massively parallel reporter assays (MPRAs) and self-transcribing active regulatory region sequencing (STARR-seq) have revolutionized enhancer characterization by enabling high-throughput functional assessment of regulatory sequences. Here, we systematically evaluated six MPRA and STARR-seq datasets generated in the human K562 cell line and found substantial inconsistencies in enhancer calls from different labs that are primarily due to technical variations in data processing and experimental workflows. To address these variations, we implemented a uniform enhancer call pipeline, which significantly improved cross-assay agreement. While increasing sequence overlap thresholds enhanced concordance in STARR-seq assays, cross-assay consistency in LentiMPRA was strongly influenced by assay-specific factors. Notably, our results show that LentiMPRA exhibits a strong preference for promoter-associated sequences rather than enhancers. Functional validation using candidate cis-regulatory elements (cCREs) confirmed that epigenomic features such as chromatin accessibility and histone modifications are strong predictors of enhancer activity. Importantly, our study validated transcription as a critical hallmark of active enhancers, demonstrating that highly transcribed regions exhibit significantly higher active rates across assays. Furthermore, we show that transcription enhances the predictive power of epigenomic features, enabling more accurate and refined enhancer annotation. Our study provides a comprehensive framework for integrating different enhancer datasets and underscores the importance of accounting for assay-specific biases when interpreting enhancer activity. These findings refine enhancer identification using massively parallel reporter assays and improve the functional annotation of the human genome.

genomics↗

bioRxiv2025

Simultaneous measurement of intrinsic promoter and enhancer potential reveals principles of functional duality and regulatory reciprocity

Accumulating evidence indicates that both promoters and enhancers are capable of exerting promoter and enhancer functions; however, the relationship between these two activities within individual elements and the determinants of this dual functionality remain poorly understood. We developed a massively parallel dual reporter assay that enables simultaneous assessment of the intrinsic promoter and enhancer potential exerted by the same sequence. Parallel quantification for thousands of elements reveals that canonical human promoters and enhancers can act as both promoters and enhancers under the same contexts, and that promoter activity may be necessary but not sufficient for enhancer function. Perturbations to element transcription factor binding motifs lead to disruptions in both activities, implicating a shared syntax for the two regulatory functions. Combinations of elements with different minimal promoters reveal reciprocal activity modulation, which, together with a strong correlation between promoter and enhancer functions, imply a bidirectional feedback loop to sustain environments of high transcriptional activity. Finally, we validate this reciprocity and correlation in situ using CRISPR activation at the human {beta}-globin locus. Our approach reveals that the functional convergence between promoters and enhancers arises from a shared regulatory logic and sequence syntax, advancing a unified model for regulatory element biology.

genomics↗

bioRxiv2024

A lineage-specific nascent RNA assay unveils principles of gene regulation in tissue biology

Gene regulatory mechanisms that modulate RNA Polymerase II activity are difficult to access in mammalian tissues composed of multiple cell lineages. Here, we develop a nascent RNA assay (PReCIS-seq) that measures lineage-specific transcriptionally-engaged Pol II on genes and DNA enhancer elements in intact mouse tissue. By employing keratinocytes as a prototype lineage, we unearth Pol II promoter-recruitment versus pause-release mechanisms operating in adult skin homeostasis. Moreover, we relate active enhancer proximity and transcription factor binding motifs on promoters to Pol II activity and promoter-proximal pausing level. Finally, we find Pol II firing rapidly into elongation on lineage identity genes and highly paused on cellular safeguarding genes in a context-dependent manner. Our work provides a basic platform to investigate mechanistic principles of gene regulation in individual lineages of complex mammalian tissues.

genomics↗

bioRxiv2023

Nanoscale photobiotinylation, pulldown and sequencing of region-specific DNA from intact cells.

Femto-seq is a novel nanoscale optical method that can be used to obtain DNA sequence information from targeted regions around a specific locus or other nuclear regions of interest. Two-photon excitation is used to photobiotinylate femtoliter volumes of chromatin within the nucleus, allowing for subsequent isolation and sequencing of DNA, and bioinformatic mapping of any nuclear region of interest in a select set of cells from a heterogenous population.

genomics↗

bioRxiv2021

Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide

Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks1,2; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification. We also introduced a new analytical tool, Peak Identifier for Nascent-Transcript Sequencing (PINTS), to identify active promoters and enhancers genome-wide and pinpoint the precise location of the 5 transcription start sites (TSSs) within these regulatory elements. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected eRNA TSSs available in 120 cell and tissue types. To facilitate the exploration and prioritization of these enhancer candidates, we also built a user-friendly web server (https://pints.yulab.org) for the compendium with various additional genomic and epigenomic annotations. With the knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the road for selection and characterization of their functions in a time-, labor-, and cost-effective manner in the future.

genomics↗

bioRxiv2020

An Improved 4'-Aminomethyltroxsalen-Based DNA Crosslinker for Biotinylation of DNA

Nucleic acid crosslinkers that covalently join commentary strands have applications as both pharmaceuticals and biochemical probes. Psoralen is a popular crosslinker moiety that reacts with double stranded DNA and RNA upon irradiation with long wave UV light. A commercially available compound EZ-Link Psoralen-PEG3-Biotin has been used in many studies to crosslink DNA and double strand RNA for genome-wide investigations. Here we present a novel probe, AP3B, which uses a psoralen derivative, 4-aminomethyltrioxsalen, to biotinylate nucleic acids. We show that this compound is 8-fold more effective at labeling DNA in cells and several hundred-fold more effective at crosslinking two strands of DNA in vitro than the commercially available compound EZ-Link Psoralen-PEG3-Biotin.

biochemistry↗

bioRxiv2020

Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins. KEY POINTSO_LIA simple to use RNA aptamer method for super-resolution STORM and PAINT imaging in cells expressing common protein tags. C_LIO_LIThe method has a number of unique advantages for live cell imaging at the nanometer scale. C_LIO_LIProvides a means to quantify the number of proteins being trafficked on the plasma membrane. C_LI

biophysics↗

bioRxiv2019

Transcription imparts architecture, function, and logic to enhancer units

Distal enhancers remain one of the least understood regulatory elements with pivotal roles in development and disease. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites (TSSs) are robust predictors of enhancer activity with higher resolution and specificity than histone modifications. We show that active enhancer units are precisely delineated by active TSSs, validate that these boundaries are sufficient to capture enhancer function, and confirm that core promoter sequences are required for this activity. Finally, we assay pairs of adjacent units and find that their cumulative activity is best predicted by the strongest unit within the pair. Synthetic fusions of enhancer units demonstrate that adjacency imposes winner-takes-all logic, revealing a simple design for a maximum-activity filter of enhancer unit outputs. Together, our results define fundamental enhancer units and a principle of non-cooperativity between adjacent units.

genomics↗

bioRxiv2019

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at heat shock loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments, topologically-associated domains (TADs), and looping interactions all remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. However, the HSF1 binding sites and the target promoters did not exhibit a focal increase in contact frequencies compared with surrounding regions, nor did we find evidence of increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the enhancer, and contact frequency with a target promoter in the non-heat shock (NHS) condition accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is pre-established in normal (uninduced) cells of diverse metazoan species.

molecular biology↗

bioRxiv2018

Glucocorticoid receptor gene polymorhisms and breast cancer: significant association in cancerous tissue samples from Turkish patients

Background and Aims: Glucocorticoid receptor (GR) has been postulated to serve an important role in normal breast and breast carcinoma. On the other hand, studies on its common polymorphisms with breast cancer are very limited. In this study, we aimed to assess the presence and frequency of 4 common polymorphisms of the GR gene in the cancerous tissue samples from breast cancer patients in the Turkish population and compare them with healthy individuals. Methods: DNA samples from 86 Turkish female breast cancer patients and 86 healthy controls analysed for BclI, Tth111I, N363S and ER22/23EK polymorphisms of GR gene by PCR-RFLP. Results: N363S polymorphism was not observed in study samples. Frequency of BclI polymorphism did not differ between cases and controls but it associated with family history of cancer. The polymorphic allele frequency of Tth111I and ER22/23EK was found to be higher in cases compared to healthy samples (p<0.05). Also, a haplotype containing polymorphic variants of these two polymorphisms found to be associated with breast cancer. Conclusion: The data presented here suggest that polymorphisms of GR gene may be an important risk factor for breast cancer predisposition.

cancer biology↗